Dihydrofolate reductase which has been purified in quantity from a methotrexate-resistant strain of Streptococcus faecium, will be used to complete determination of the primary sequence, to determine additional residues involved in substrate binding and/or catalysis, and to produce crystals more suitable for X-ray analysis of structure than those available. Beef liver dihydrofolate reductase will be purified in quantity by a method already devised and used for studies similar to those indicated for the bacterial enzyme. Techniques and equipment acquired for sequence determination on the bacterial enzyme will be employed for the beef liver enzyme. Special attention will be directed towards crystallization of the beef liver enzyme since preliminary examination indicates that it is more stable under conditions suitable for crystallization than the bacterial enzyme. The reductase from a human leukemic cell line now being maintained in the laboratory will be examined in a preliminary fashion. A purification procedure similar to that used for the beef liver will be examined for efficiency of recovery and for applicability on a large scale. Information from these studies will be used to elucidate the mechanism of dehydrogenase action, the mechanism of very tight binding of inhibitors, of methotrexate resistance, and of inhibitor selectivity.